Esterase free enzymes

ABSTRACT

A process for decreasing esterase activity present in the presence of D-amino acid oxidase activity or in the presence of glutarylacylase activity in a mixture having esterase activity and D-amino acid oxidase activity and/or having esterase activity and glutarylacylase activity by treatment with phenylmethylsulphonyl fluoride; immobilized biocatalyts in spherical particle form obtainable by treating microorganism cells having enzyme activity with a primary or secondary amine containing polymer, an organic solvent which is able to form a two-phase system with water and a bifunctional agent; and the use of that process and biocatalysts in the production of  7 -aminocephalo-sporanic acid,  7 -amino- 3 -hydroxymethyl- 3 -cephem- 4 -carboxylic acid and cephalosporin antibiotics.

[0001] The present invention relates to a process for inhibition ofesterase activity in enzyme preparations, e.g. useful in enzymaticcatalysed production of 7-aminocephalosporanic acid (7-ACA); and to theproduction of enzyme preparations, e.g. useful in 7-ACA and/or7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid production.

[0002] 7-ACA is a key intermediate in the production of e.g.pharmaceutically, active cephalosporin antibiotics, e.g. 7-ACA may beacylated at the amine group in position 7 of the ring system, e.g. agroup known as a valuable group in the production of cephalosporinantibiotics or known as a valuable group in an intermediate for theirproduction; e.g. to obtain a 7-aminoacylated cephalosporin having amethylacetoxy group in position 3 of the ring system, such ascefotaxime, cefpodoxime (proxetil), cephaloglycin; and in the productionof other cephalosporin antibiotics or intermediates for their productionwhich may be derived from 7-ACA; e.g. by further reacting themethylacetoxy group in position 3 of the ring system to obtain a3-substituted cephalosporin substituted by a group which is differentfrom the acetoxymethyl group; e.g. a group known as a valuable group inthe production of cephalosporin antibiotics or known as a valuable groupin an intermediate for their production; e.g. by nucleophilicsubstitution of the acetoxy group, or e.g. by deacetylation of themethylacetoxy group to obtain the hydroxymethyl group (e.g. HACA); ande.g. further reacting a hydroxymethyl group obtained in position 3 ofthe ring system, e.g. by nucleophilic substitution of the hydroxy group;or removal of the hydroxy function of a hydroxymethyl group or removalof the hydroxmethyl group in position 3 of the ring system; and, ifdesired, esterification of the carboxylic group in position 4 of thering system, e.g. by a group known as a valuable group in the productionof cephalosporin antibiotics or known as a valuable group in anintermediate for their production; and, if desired, salt and/or solventformation of a cephalosporin compound obtained in such a reaction e.g.according to a conventional method. 7-ACA and HACA may e.g. be obtainedfrom cephalosporin C (Ceph C) by deacylation of the amine group inposition 7 of the ring system, and deacetylation of the methylacetoxygroup in position 3of the ring system, respectively, e.g. enzymatically.An enzyme useful in enzymatic Ceph C deacylation is e.g. D-amino acidoxidase (DAO) which catalyses the oxydative desamination of Ceph C toform glutaryl-7-aminocephalosporanic acid (GI-7-ACA) via theintermediate α-ketoadipoyl-7-aminocephalosporanic acid (KA-7-ACA).GI-7-ACA may be hydrolised to obtain glutaric acid and 7-ACA, e.g. by aglutarylacylase (GAC). It is known that DAO and GAC containing enzymepreparations, such as microorganism cells may contain esterase activityadditionally. Undesired deacetylation of the side chain in position 3 ofthe ring structure of the cephalosporin may occur, e.g. due to thepresence of esterase activity in enzymatically catalysed Ceph Cdeacylation e.g. 3-hydroxymethyl derivatives of Ceph C and GI-7-ACA maybe formed. This may result in a significant decrease of 7-ACA qualityand yield. Conventional methods for decreasing esterase activity presentin the presence of DAO activity by acetone or CUSO₄ treatment maydecrease esterase activity incompletely.

[0003] It was now surprisingly found that the treatment of a mixturehaving esterase activity in the presence of DAO activity or havingesterase activity in the presence of GAC activity withphenylmethylsulphonyl fluoride (PMSF) may decrease esterase activityconsiderably, e.g. substantially complete, whereas DAO, or GAC activity,respectively may remain high, e.g. substantially unchanged. This findingis surprising because according to the present invention PMSF, knowne.g. as an irreversible inhibitor of serine containing proteins byserine sulphonylation, may decrease esterase activity present in thepresence of DAO activity or in the presence of GAC activity selectivelywithout, e.g. substantial influence on DAO or GAC activity; and evenmore surprising is the selective and effective inhibition by PMSF ofesterase activity present in the presence of GAC activity, e.g. becauseof similar function and structure of acylases and esterases.

[0004] In one aspect the present invention provides a process fordecreasing, e.g. substantially removing esterase activity present in thepresence of D-amino acid oxidase activity or in the presence ofglutarylacylase activity in a mixture having esterase activity andD-amino acid oxidase activity and/or glutarylacylase activity, e.g.present in the form of microorganism cells or in the form of a cell-freeextract thereof, e.g. in the form of a cell-free extract thereof,comprising treating a mixture having esterase activity and D-amino acidoxidase activity and/or glutarylacylase activity withphenylmethylsulphonyl fluoride, e.g. wherein D-amino acid oxidaseactivity or glutarylacylase activity remains, e.g. substantially thesame after treatment with phenylmethylsulphonyl fluoride as before saidtreatment. Typically D-amino acid oxidase activity remains more than 91%of the original value and in the case of glutarylacylase activity evenmore than 97% of the original value.

[0005] A process of the present invention may e.g. be carried out asfollows:

[0006] Known and e.g. commercially available microorgansisms producingDAO activity include e.g. Trigonopsis, Aspergillus, Penicillium,preferably Trigonopsis variabilis microorganisms. Known and e.g.commercially available microorgansisms producing GAC activity includee.g Pseudomonas, Achromobacter, Bacillus cereus or e.g. transformants,e.g. E. coli transformants, e.g. transformed according to e.g. aconventional method. A mixture having DAO activity or GAC activity andesterase activity may e.g. be obtained commercially or e.g. according toa conventional method, and may be e.g. in the form of a cell-freeextract, e.g. in immobilised form, or in the form of microorganismcells, such as in the form of e.g. partly purified or impurified cells,and/or permeabilised or non-permeabilised cells, and/or partly destroyedor intact cells, and/or immobilised or non-immobilised cells; such asobtainable from a fermentation broth, e.g. according to a conventionalmethod, preferably in the form of a cell-free extract, e.g. immobilized.E.g. microorganism cells may be isolated, e.g. harvested from afermentation broth and used as such, e.g. in moist form, e.g. aftercentrifugation of the fermentation broth, or the cells may be furthertreated before or after isolation from the fermentation broth, e.g.according to a conventional method, such as homogenising cells toobtain, e.g. partly, destroyed cells, and/or permeabilising cells orfragments thereof to obtain permeabilised cells or cell fragments,and/or purifying cells or cell fragments to obtain e.g. partly purifiedcells and/or cell fragments and/or cell-free (e.g. by cell flocculation)extracts, and/or immobilising cells or cell-free extracts to obtainimmobilised cells and/or immobilised cell fragments and/or immobilisedcell-free extracts. E.g. immobilisation may be carried out according toa conventional method; e.g. in the presence of acrylic (immobilisation)beads, such as Eupergit^(R) or in the presence of an ion exchange resin,e.g. such as Relite Dianion^(R) according to a method described in theexamples below; or e.g. according to another aspect of the presentinvention which is described below.

[0007] The microorganism cells may be used in the form of an, e.g.buffered, aqueous cell suspension, obtainable e.g. by re-suspension ofcells in water or in a buffer solution after isolation from afermentation broth, or in case of a cell-free extract an aqueoussolution of the cell-free extract may be used. The pH of a suspension orsolution may be, e.g. approximately neutral, e.g. a pH of 6.5 to 8.5,such as 7.0 or around 7.0 may be appropriate.

[0008] In a preferred embodiment of the present invention an esteraseand DAO activity having mixture may be e.g. substantially, free of e.g.native catalase activity, e.g. obtainable by treatment of an aqueouscell mixture having DAO, esterase and catalase activity in basic medium,e.g. in the presence of a caustic soda solution, e.g. at pH 9 to 11.5,such as 10 to 11.

[0009] An esterase activity and DAO activity having mixture or anesterase activity and GAC activity having mixture, may be treated, e.g.incubated at an appropriate temperature, e.g. at room temperature for anappropriate time, e.g. for several hours, such as for 1 to 5, e.g. 2.5to 4 hours or longer with PMSF, e.g. by addition of a PMSF solution,e.g. of an appropriate concentration range, e.g. of 0.5% to 25%, such as1% to 10% PMSF in an appropriate solvent, e.g. in alcohol, such as(C₁₋₄)alkanol, e.g. ethanol. Per 100 liters of a cell suspension anamount of e.g. 1000 ml of PMSF solution, e.g. in a concentration rangeas indicated above, may be sufficient to remove practically completelyand irreversibly undesired esterase activity, e.g. without, e.g.substantially decreasing the DAO or GAC activity present in the startingmixture. Removal of esterase activity may be determined by determinationof deacetylation products obtained in GI-7-ACA or 7-ACA reaction fromCeph C using a PMSF-treated DAO or GAC containing mixture. Forcomparison the reaction may be carried out using a non-PMSF treated DAOor GAC containing mixture under comparable reaction conditions.

[0010] A mixture may be obtained wherein DAO or GAC activity is high,e.g. substantially the same as before PMSF treatment and whereinesterase activity is, e.g. substantially, completely removed. A mixtureobtainable according to the present invention is useful, e.g. in theproduction of 7-ACA from e.g. Ceph C. Yields and purity of 7-ACAobtained may be improved if PMSF treated DAO and/or GAC activity is usedin comparison with the use of non-PMSF treated DAO and/or GAC activityunder comparable reaction conditions. Thus, e.g. the removal of esteraseactivity according to the present invention, results in less, e.g.substantially no, 3-deacetylation by-products which are due to esteraseactivity during DAO and or GAC reaction. The undesired amount of3-deacetylation by-products in GI-7-ACA or 7-ACA obtained by a processaccording to the present invention may be, e.g. substantially (typicallywithin 1% in a process giving 3% starting activity whereas when PMSF isnot used it may increase to 6%), the same as in Ceph C used as startingmaterial.

[0011] In another aspect the present invention provides a process forthe production of glutaryl-7-aminocephalosporanic acid comprising thesteps

[0012] i) treating a mixture having esterase activity and D-amino acidoxidase activity with phenylmethylsulphonyl fluoride, e.g. to obtain amixture which is substantially free of esterase activity, and e.g.having substantially the same D-amino acid oxidase activity as beforePMSF treatment,

[0013] ii) reacting cephalosporin C with a mixture obtained in step i)to obtain glutaryl-7-aminocephalosporanicacid, and isolatingglutaryl-7-aminocephalosporanic acid, if desired.

[0014] In another aspect the present invention provides a process forthe production of 7-aminocephalosporanic acid comprising the steps

[0015] i) treating a mixture having esterase activity and D-amino acidoxidase activity with phenylmethylsulphonyl fluoride, e.g. to obtain amixture which is substantially free of esterase activity, and e.g.having substantially the same D-amino acid oxidase activity as beforePMSF treatment,

[0016] ii) reacting cephalosporin C with a mixture obtained in step i)to obtain glutaryl-7-aminocephalosporanic acid,

[0017] iii) converting glutaryl-7-aminocephalosporanic acid into7-aminocephalosporanic acid, and isolating 7-aminocephalosporanic acidobtained, if desired. Conversion of glutaryl-7-aminocephalosporanic acidinto 7-aminocephalosporanic acid may be cariied out according to aconventional method, e.g. chemically or enzymatically, or e.g. accordingto another aspect of the present invention, e.g. as described herein.

[0018] In another aspect the present invention provides a process forthe production of 7-amino cephalosporanic acid comprising the steps

[0019] i) treating a mixture having esterase activity and D-amino acidoxidase activity with phenylmethylsulphonyl fluoride, e.g. to obtain amixture which is substantially free of esterase activity, and e.g.having substantially the same D-amino acid oxidase activity as beforePMSF treatment,

[0020] ii) reacting cephalosporin C with a mixture obtained in step i)to obtain glutaryl-7-aminocephalosporanic acid,

[0021] iii) treating a mixture having esterase activity andglutarylacylase activity with phenylmethylsulphonyl fluoride, e.g. toobtain a mixture which is substantially free of esterase activity, ande.g. having substantially the same glutarylacylase activity as beforePMSF treatment,

[0022] iv) reacting glutaryl-7-aminocephalosporanic acid obtained instep ii) with a mixture obtained in step iii) to obtain7-aminocephalosporanic acid; and isolating 7-aminocephalosporanic acidobtained in step ii), if desired.

[0023] A process of the present invention to obtain 7-ACA from Ceph Cmay e.g. be carried out as follows: The starting material Ceph C may bee.g. in the form of an aqueous suspension or solution of Ceph C infree-form or in salt form; e.g. Ceph C may be e.g. in the form of a CephC containing fermentation broth, e.g. wherein cells and solid have beenremoved, e.g. according to a conventional method, or in the form of asalt of Ceph C, e.g. in the form of a sodium salt. The suspension orsolution of Ceph C may be treated with a mixture having DAO activitywhich is pre-treated with PMSF according to the present invention, e.g.in a form as described above, e.g. an aqueous microorganism cellsuspension or a cell-free extract may be added to an aqueous suspensionor solution of Ceph C (or vice versa); under introduction of oxygen intothe suspension or solution, e.g. in the form of oxygen gas or in theform of air, or mixtures thereof, e.g. if desired under pressure.

[0024] The reaction may be carried out at appropriate pH, e.g. at aroundneutral pH, e.g. at a pH of between 6.5 to 8.0, such as 7.0 to 7.5, atappropriate temperatures such as 10° C. to 30° C., e.g. at roomtemperature. The amount of DAO activity, e.g. the amount of a cellsuspension or of a cell-free extract having DAO activity in respect withthe amount of Ceph C is depending on the DAO activity present-in thecell suspension used and on the amount of Ceph C to be reacted and isnot critical; an amount e.g. necessary for a short reaction time may beeasily determined e.g. by determination of the amount of GI-7-ACA formedin the mixture during, e.g. regular time intervalls by a conventionalmethod, e.g. by HPLC determination.

[0025] GI-7-ACA obtained may be isolated and purified, e.g. by aconventional method or may be used as such for further processing, e.g.in the production of 7-ACA.

[0026] Oxidase activity may be determined as follows: 0.2 to 5 g of amixture having DAO activity in the form of permeabilised cells issuspended under stirring in an aqueous 20 mM Ceph C solution at pH 8.0,at 25° C. under introduction of an of oxygen stream into the mixture tooxygen saturation.

[0027] The increase of GI-7-ACA and KA-7-ACA is determined by HPLC. Theactivity of “1 U oxidase” corresponds to the formation of 1 μmol/minGI-7-ACA and KA-7-ACA under the above conditions.

[0028] An aqueous solution or suspension of GI-7-ACA, e.g. obtainable orobtained according to the present invention may be brought into contactwith a mixture having GAC activity which is pretreated with PMSFaccording to the present invention, e.g. an aqueous, e.g. buffered cellsuspension or a cell-free extract may be added to an aqueous suspensionor solution of GI-7-ACA (or vice versa). The reaction may be carried outat appropriate pH, e.g. at slightly basic pH, e.g. including an pH ofbetween 7.5 to 8.5, such as around 8.2, at appropriate temperaturesincluding 10° C. to 30° C., e.g. room temperature. The amount of GACactivity in respect with the amount of GI-7-ACA is depending on the GACactivity present in the cell suspension or in the cell-free extract usedand on the amount of GI-7-ACA to be reacted and is not critical; anamount e.g. necessary for a short reaction time may be easily determinede.g. by determination of the amount of 7-ACA formed in the mixtureduring, e.g. regular time intervalls by a conventional method, e.g. byHPLC determination.

[0029] GAC activity may be determined as follows: 0.5 g to 2.0 g of amixture having GAC activity are suspended under stirring in a 2% aqueousGI-7-ACA solution containing 5 mM phosphate and of a pH of 8.0 at 37° C.The pH is kept at 8.0 by addition of an aqueous 100 mM sodium hydroxidesolution. The amount of sodium hydroxide used is determined andcorresponds to the amount of glutaric acid or 7-ACA, respectively formedfrom GI-7-ACA. The activity of “1 U acylase” corresponds to theformation of 1 μmol/min glutaric acid or 7-ACA, respectively under theabove conditions.

[0030] 7-ACA obtained may be isolated and purified, e.g. by aconventional method, or, if desired may be used as such, e.g. forfurther processing, e.g. in a 7-ACA reaction, e.g. according to aconventional method.

[0031] 7-ACA obtained according to the present invention may be of highpurity, e.g. 7-ACA obtained may contain a low amount of 3-deacetylationproducts and may be obtained in high yields; e.g. in yields which arehigher than in a process wherein a non pretreated DAO activity and/or anon-pre-treated GAC activity is used under comparable reactionconditions.

[0032] It is known that e.g. enzyme containing microorganism cells maybe immobilised. Immobilised enzyme containing microorganism cells may beuseful e.g. because of the possibility of easy recovering and re-use.Known processes for the production of immobilized microorgansim cellsmay have disadvantages, e.g. immobilised cells having poor enzymestability and/or low specific enzyme activity may be obtained, and/orthe immobilisation process may be complicated. Surprisingly a simpleimmobilisation process for microorganism cells having an enzymeactivity, such as DAO or GAC activity, was now found wherein immobilisedcells may be obtained in the form of solid spherical particles which maycontain stable and highly specific enzyme activity.

[0033] In another aspect the present invention provides a process forthe production of spherical particles, e.g. in solid form, frommicroogranism cells having an enzyme activity, comprising the steps

[0034] i) treating microorganism cells with a primary or secondary aminecontaining polymer,

[0035] ii) contacting an organic solvent which is able to form atwo-phase system with water with a mixture of microorganism cells, e.g.adding a mixture of microorganism cells to a solvent which is able toform a two phase system with water,

[0036] iii) treating a mixture obtained in step ii) with a bifunctionalagent; and isolating the spherical particles obtained;

[0037] e.g. further comprising

[0038] pretreating the microorganism cells with a bifunctional reagentbefore carrying out steps i) or ii); and/or

[0039] adding a water-miscible solvent to a mixture obtained in stepiii) before isolation of the spherical particles; and/or

[0040] adding a solid to a mixture obtained in step i) or to themicroorganism cells before carrying out step i).

[0041] The process of the present invention is useful for immobilisingmicroorganism cells having an enzyme activity which are commonlydesignated as biocatalysts. Microorganisms which may be used accordingto the present invention include all types of microorganisms, such asbacterias, yeasts, funghi and actinomycetes, e.g. Trigonopsis, such asTrigonopsis variabilis, Agrobacterium, such as Agrobacteriumradiobacter, Rhodotorula, such as Rhodotorula glutinis, Pleurotus, suchas Pleurotus ostreatus, Aspergillus, Penicillium, Pseudomonas,Achromobacter, Bacillus, such as Bacillus cereus, Schizosaccharomyces,such as Schizosaccharomyces pombe; or e.g. transformants such as e.g.Escherichia coli transformants having an enzyme activity. Enzymeactivity includes all types of enzyme activity, such as hydrolase,isomerase, lyase, decarboxylase, oxireductase activity. Examples ofmicroorganism cells having an enzyme activity includes e.g. Pleurotus,e.g. Pleurotus ostreatus cells having e.g. penicillin-V acylaseactivity, Agrobacterium, e.g. Agrobacterium radiobacter cells havinge.g. hydantoinase and/or N-decarbamoylase activity, Rhodotorula, e.g.Rhodotorula glutinis cells having e.g. esterase and/or oxidase activity,Trigonopsis, e.g. Trigonopsis variabilis having e.g. DAO activity,Schizosaccharomyces, such as Schizosaccharomyces pombe having e.g. GACactivity; and cells of transformants such as Escherichia coli havinge.g. GAC and/or esterase activity.

[0042] The process of the present invention may be e.g. of particularinterest in the production of immobilised microorganism cells which areuseful in 7-ACA or HACA production, e.g. starting from Ceph C, such ascells having DAO activity, e.g. in the presence of esterase (and/ordeacetylase) activity, and/or cells having GAC activity, e.g. in thepresence of esterase (and/or deacetylase) activity.

[0043] The process according to the present invention may be carried outas follows:

[0044] Microorganism cells having enzyme activity may e.g. be obtainedfrom a fermentation broth, e.g. according to a conventional method.Microorganism cells include cells in any form e.g. in the form of, e.g.partly purified or impurified cells, and/or permeabilised ornon-permeabilised cells, and/or, e.g. partly, destroyed or intact cells.E.g. microorganism cells may be isolated, e.g. harvested from afermentation broth and used as such, e.g. in moist form, e.g. aftercentrifugation of the fermentation broth, or the cells may be furthertreated before or after isolation from the fermentation broth, e.g.according to a conventional method, such as homogenising cells toobtain, e.g. partly, destroyed cells and/or permeabilising cells orfragments thereof to obtain permeabilised cells or fragments thereof,and/or purifying cells to obtain e.g. partly purified cells and/or cellfragments.

[0045] Undesired enzyme activity in microogrganism cells may be removedbefore immobilisation of the cells, e.g. according to a conventionalmethod. E.g. catalase activity in Trigonopsis, e.g. Trigonopsisvariabils cells may e.g. be removed by treatment under a basic pH, e.g.a pH of 9 to 12, e.g. by addition of a caustic soda solution, e.g. in amixture of microorganism cells alone, or in a mixture of microorganismcells in the presence of a primary and/or secondary amine containingpolymer, optionally in the presence of additives, such asβ-mercaptoethanol, ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA) forsome time, e.g. ca. 15 minutes or longer.

[0046] According to the present invention the microorganism cells may bepre-treated with a bifunctional agent. A bifunctional agent as usedherein is to be understood as a compound having at least two reactivegroups which are able to react with primary and/or secondary aminegroups, including e.g. glutaraldehyde, dimethyl pimelimidate,epichlorhydrin, N,N′-carbonyldimidazole, 1,4-butanediol-diglycidylether,maleic acid anhydride, dicyclohexylcarbodiimide,hexamethylenediisocyanate, preferably glutaraldehyde. Pre-treatment ofthe cells may be carried out in the fermentation broth, or afterisolation of microorganism cells, preferably in the fermentation broth.E.g. the bifunctional agent may be added to a microorganism cellsuspension, e.g. in dissolved form, e.g. glutaraldehyde may be used inaqueous solution, e.g. in 10% to 30% aqueous solution and the mixtureobtained after addition may be stirred for some time, e.g. 30 minutes tosome hours, such as 30 minutes to S hours. The amount of thebifunctional agent in respect with the amount of cells is not critical;e.g. per 100 g of dry cell weight e.g. 0.1 g to 100 g, e.g. 5 g to 50 gof a bifunctional agent may be used conveniently; e.g. if glutaraldehydeis used as a bifunctional agent 0.5 g to 200 g, such as 1 g to 100 g ofa 25% aqueous solution may be appropriate. After pre-treatment with abifunctional agent the cells may be harvested and isolated, e.g.according to a conventional method, such as concentrated bymicrofiltration and/or centrifugation and stored in a cool place, e.g.in frozen state. For further use frozen cells may be thawed and washedwith spring water, and, if desired, concentrated, e.g. according to aconventional method such as microfiltration.

[0047] The microorganism cells, e.g. pre-treated as described above, maybe used in the form of an, e.g. buffered, aqueous cell suspension e.g.by re-suspenion of cells in water after isolation from a fermentationbroth and optional further treatment, e.g. at a pH of 6.0 to 10.0, suchas 8.0 or around 8.0. If desired, an additive, e.g. as conventional,such as β-mercaptoethanol, ethylenediamine-N,N,N′,N′-tetraacetic acid(EDTA) may be added to the suspension, e.g. in a conventional amount. Tothe suspension obtained a primary and/or secondary amine containingpolymer including e.g. a polyethylene imine and a polyvinylamine, whichis preferably water-soluble, e.g. in aqueous solution, such aspolyethylene imine in e.g. 10% to 50% aqueous solution, e.g. having amolecular weight of 600,000 to 1,000,000 is added. Primary and/orsecondary amine containing polymers are e.g. commercially available ore.g. may be produced according to a conventional method. The amount of aprimary and/or secondary amine containing polymer in respect with thedry cell mass is not critical and includes e.g. an amount of 1 g to 100g, such as 5 g to 70 g, such as 10 g to 50 g per 100 g of drymicroorganism cell weight. The cell/polymer mixture is contacted with anorganic solvent which is able to form a two-phase system with water,e.g. the cell/polymer mixture is introduced into an organic solventwhich is able to form a two-phase system with water. Appropriate organicsolvents include e.g. trialkyl phosphates, such as tributyl phosphates,e.g. n-tributyl phosphate, alkanes, e.g. n-hexane, n-heptane, aromatichydrocarbons, e.g. toluene, natural or synthetic oils, e.g. soya oil,silicone oil, diesel oil, aromatic or aliphatic e.g. (C₃₋₈)alkyl, suchas (C₄₋₆)alkyl, e.g. mono- or di-carboxylic acid alkyl, e.g.(C₁₋₈)alkyl, such as (C₁₋₄)alkyl, including e.g. glutaric, succinic,adipic acid dimethyl- or diethylester, benzoic or phthalic acid ethyl-,butyl-, dibutyl- or diisobutylester and anise alcohol, preferablytrialkyl phosphate. The amount of organic solvent may be such that themicroorganism cell supension may be dispersed in the organic solvent,e.g. by, e.g. laminar, stirring into the solvent; per g of dry cellweight e.g. 1 ml to 100 ml, such as 3 ml to 50 ml of solvent mayconveniently be used.

[0048] The mixture containing the cells, the polymer and the organicsolvent is stirred for some time, e.g. until a homogeneous supension isobtained and treated with a bifunctional agent, including e.g.glutaraldehyde, epichlorohydrin, N,N′-carbonyldiimidazole,1,4-butanediol-diglycidylether, maleic acid anhydride,dicyclohexylcarbodiimide, hexamethylenediisocyanate, preferablyglutaraldehyde. If a pre-treatment of the microorganism cells with abifunctional agent was carried out, the same bifunctional agent mayconveniently be used as in the pre-treatment. The amount of thebifunctional agent is not critical and includes an amount of e.g. 0.5 gto 100 g of a bifunctional agent per 100 g of dry cell weight, e.g. incase of using glutaraldehyde as a bifunctional agent e.g. 1 g to 200 gof a 25% aqueous solution may conveniently be used. Upon addition of thebifunctional agent to the mixture containing the cells, the polymer andthe organic solvent crosslinking may start and may be terminated e.g.within a few minutes and a suspension of, e.g. solid spherical particlesof the microorganism cells in the solvent mixture used may be obtained.If desired, the suspension obtained may be contacted with an organicsolvent which is water-miscible and which may be harmless in respectwith enzyme activity, e.g. which may be non-damaging in respect withenzyme activity, including e.g. alcohols, ketones, polyethylene glycols,glycerol, preferably glycerol, e.g. the cell suspension obtained may beintroduced into the organic solvent or the solvent may be introducedinto the cell suspension obtained. The amount of the water-miscibleorganic solvent is not critical and includes an amount which issufficient to form a liquid-liquid two phase system in the reactionmixture; e.g. per 100 g of dry cell weight conveniently e.g. 100 g to5000 g and more, e.g. 500 g to 5000 g of a water-miscible organicsolvent may be used. Phase separation, e.g. immediate, may occur uponcontact of the solvent with the cell suspension. Spherical cellparticles, e.g. solid, formed may be in the lower aqueous phaseobtained. The upper phase, e.g. containing mainly the organic solventwhich is able to form a two phase system with water may be separatedeasily and, e.g. substantially completely from the lower phase and maybe re-used, e.g. in a further immobilisation process. The spherical cellparticles obtained may be in solid form and may be isolated form theorganic-miscible-solvent phase, e.g. by a conventional method, e.g. bypouring the suspension through a vessel with perforated bottom,centrifugation, filtration. The isolated spherical particles may befreed from solvent residues, e.g. washed, e.g. with spring water.

[0049] Prior to crosslinking, i.e. prior to addition of the bifunctionalagent, e.g. prior to addition of a solvent which is able to form atwo-phase system with water, a solid, including e.g. aluminium oxide,activated carbon, bentonite may be added to the mixture of cells. Theamount of a solid is not critical; conveniently an amount of 5 g to 200g, such as 50 g to 150 g per 100 g dry cell weight may be used. Aftersuspension in the solvent which is able to form a two phase system withwater and crosslinking by addition of the bifunctional agent into such amixture, spherical cell particles may be obtained which may particularlyeasy be separated and which may have a favourable narrowsize-distribution-range and excellent mechanical stability.

[0050] The biocatalyst, e.g. spherical microorganism cell particles,e.g. in solid form, obtained according to the present invention may bestored for later use e.g. in a cool place, e.g. in an appropriate bufferor e.g. in frozen form or lyophilised form.

[0051] Spherical particles containing microorganism cells which containenzyme activity obtainable, e.g. obtained, according to the presentinvention are novel.

[0052] In another aspect the present invention provides sphericalparticles containing microorganism cells, e.g. in solid form, whichcontain enzyme activity obtainable, e.g. obtained, from a processcomprising the steps

[0053] i) treating microorganism cells having enzyme activity with aprimary or secondary amine containing polymer,

[0054] ii) contacting an organic solvent which is able to form atwo-phase system with water with a mixture of microorganism cells, e.g.after carrying out step i), e.g. by addition of a mixture ofmicroorganism cells to a solvent which is able to to form a two-phasesystem with water,

[0055] iii) treating a mixture obtained in step ii) with a bifunctionalagent; and isolating the spherical particles obtained;

[0056] e.g. further comprising

[0057] pre-treating the microorganism cells with a bifunctional reagentbefore carrying out steps i) or ii); and/or

[0058] adding a water-miscible solvent to a mixture obtained in stepiii) before isolation of the spherical particles; and/or

[0059] adding a solid to a mixture obtained in step i) or tomicroorganism cells before carrying out step i).

[0060] Spherical particles, e.g. solid, having an enzyme activityobtained according to a process of the present invention may be usefulin enzymatic reactions, e.g. spherical particles, e.g. solid, having DAOactivity, e.g. (pre-)treated with PMSF and spherical particles, e.g.solid having GAC activity, e.g. (pre-)treated with PMSF may be useful inthe production of 7-ACA and/or GI-7-ACA and/or HACA.

[0061] In another aspect the present invention provides a process forthe production of glutaryl-7-aminocephalosporanic acid comprising thesteps

[0062] i) treating microorganism cells having D-amino acid oxidaseactivity with a primary or secondary amine containing polymer,

[0063] ii) contacting an organic solvent which is able to form atwo-phase system with water with a mixture of microorganism cells, ,e.g. after carrying out step i), e.g. by addition of a mixture ofmicroorganism cells to a solvent which is able to to form a two-phasesystem with water,

[0064] iii) treating a mixture obtained in step ii) with a bifunctionalagent; and isolating the spherical particles obtained, and if desired,treating a mixture having esterase activity in the presence of D-aminoacid oxidase activity with phenylmethylsulphonyl fluoride, e.g. beforestep iii), e.g. before step i); or treating spherical particles obtainedin step iii) having esterase activity in the presence of D-amino acidoxidase activity with phenylmethylsulphonyl fluoride,

[0065] iv) reacting cephalosporin C with spherical particles obtained instep iii) to obtain glutaryl-7-aminocephalosporanic acid, and isolatingglutaryl-7-aminocephalosporanic acid, if desired;

[0066] e.g. and further converting glutaryl-7-aminocephalosporanic acidobtained in step iv) into 7-aminocephalosporanic acid.

[0067] In another aspect the present invention provides a process forthe production of glutaryl-7-aminocephalosporanic acid which comprisesreacting cephalosporin C with spherical particles obtainable by stepiii) to obtain glutaryl-7-aminocephalosporanic acid, and isolatingglutaryl-7-aminocephalosporanic acid, if desired.

[0068] Conversion of glutaryl-7-aminocephalosporanic acid obtained instep iv) into 7-aminocephalosporanic acid may e.g. be carried outaccording to a conventional method, e.g. chemically or enzymatically, oraccording to another aspect of the present invention, e.g. usingspherical particles having GAC activity obtainable or obtained asdescribed above according to the present invention. If according to thepresent invention spherical particles are used having DAO enzymeactivity in the presence of deacetylase activity; and/or having GACactivity in the presence of deacetylase activity, HACA may be obtainedin a reaction with Ceph C or GI-7-ACA.

[0069] In another aspect the present invention provides the use of aprocess for the production of spherical particles according to thepresent invention or the use of spherical particles produced accordingto the present invention in the production of7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid from cephalosporin Cor from glutaryl-7-aminocephalosporanic acid.

[0070] 7-ACA or HACA produced according to the present invention may beuseful as an intermediate in the production of a cephalosporin, e.g. acephalosporin antibiotic or another intermediate in the productionthereof, e.g. by

[0071] acylating the amine group in position 7 of the ring system

[0072] further reacting the acetoxymethyl group in position 3 of thering system

[0073] esterification of the carboxy group in position 4 of the ringsystem

[0074] salt and/or solvent formation,

[0075] e.g. as described above.

[0076] In another aspect the present invention provides a process forthe production of 7-aminocephalosporanic acid comprising the steps

[0077] i) treating microorganism cells having glutarylacylase activitywith a primary or secondary amine containing polymer,

[0078] ii) contacting an organic solvent which is able to form atwo-phase system with water with a mixture of microorganism cells,

[0079] iii) treating a mixture obtained in step ii) with a bifunctionalagent; and isolating the spherical particles obtained,

[0080] iv) if desired, treating a mixture having esterase activity inthe presence of glutarylacylase activity with phenylmethylsulphonylfluoride, e.g. before step iii), e.g. before step i) if desired; ortreating spherical particles obtained in step iii) having esteraseactivity in the presence of glutarylacylase acylase withphenylmethylsulphonyl fluoride, if desired,

[0081] v) reacting glutaryl-7-aminocephalosporanic acid with sphericalparticles obtained in step iii) or obtained in step iv) to obtain7-aminocephalosporanic acid; and isolating 7-aminocephalosporanic acidobtained, if desired.

[0082] In another aspect the present invention provides a process forthe production of a cephalosporin, e.g. a cephalosporin antibiotic or anintermediate in its production, wherein 7-aminocephalosporanic acid or7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid, e.g.7-aminocephalosporanic acid, produced according to the present inventionis used as an intermediate.

[0083] “Substantially” as used herein means e.g. greater than 95%, e.g.96%, 97% of the original value or less than 5%, e.g. 4%, 3% of theoriginal value as appropriate.

[0084] In the following examples all temperatures are given in degreesCelsius.

[0085] The following abbreviations are used:

[0086] 7-ACA: 7-aminocephalosporanic acid

[0087] Ceph C: Cephalosporin C

[0088] DAO: D-amino acid oxidase

[0089] GAC: glutarylacylase

[0090] GDA: glutaraldehyde

[0091] GI-7-ACA: Glutaryl-7-aminocephalosporanic acid

[0092] KA-7-ACA:α-ketoadipoyl-7-aminocephalosporanic acid

[0093] PEI Polyethylene imine

[0094] PMSF: Phenylmethylsulphonyl fluoride

[0095] TBP: n-Tributyl phosphate

[0096] The activity of “1 U oxidase” corresponds to the formation of 1μmol/min GI-7-ACA and KA-7-ACA in an oxygen saturated 20 mM aqueous CephC solution at pH 8 and 25° C.

[0097] The activity of “1 U acylase” corresponds to the formation of 1μmol/min glutaric acid or 7-ACA in a 2% aqueous GI-7-ACA solution at pH8 and 37° C.

EXAMPLE 1

[0098] General Procedure to Obtain a Mixture Having DAO Activity Whichis Substantially Free of Esterase Activity

[0099] From an aqueous fermentation broth having esterase activity andDAO activity in water, a cell pellet is obtained from harvested cellsaccording to a conventional method (see e.g. Kubicek-Pranz, E. M. etal., Can. J. Microbiol. 1985, 31, pp 624-628), 10 g of the cell pelletobtained are re-suspended in 50 ml of water and treated for ca. 90minutes at a pH of ca. 11 with a caustic soda solution at roomtemperature to remove catalase activity. The pH of the mixture isadjusted to ca. 7 by addition of phosphoric acid.

[0100] To the mixture obtained 500 μl of an ethanolic solutioncontaining 10 mg of PMSF/ml ethanol are added and the mixture obtainedis incubated at room temperature for ca. 3 hours. The cell suspensionobtained may be used as such in a reaction which requires DAO activity.

[0101] DAO activity of cell pellet: 530 U oxidase

[0102] DAO activity after caustic soda treatment: 490 U oxidase

[0103] DAO activity after PMSF treatment: 450 U oxidase (=92% ofactivity before PMSF treatment).

EXAMPLE 2

[0104] A PMSF-treated cell suspension having DAO activity obtained asdescribed example 1 is added to a solution of 10 g Ceph C in 1000 ml ofwater and the pH of the solution obtained is adjusted to 7.5. Themixture obtained is stirred for ca. 180 minutes at room temperatureunder introduction of oxygen into the mixture.

[0105] Yield of GI-7-ACA: 91%.

[0106] Sum of 3-deacetylation products in the starting Ceph C solution:3% relative to Ceph C

[0107] Sum of 3-deacetylation products in reaction solution obtained: 3%relative to starting Ceph C

[0108] Example 2 is repeated but using a DAO activity without PMSFtreatment Yield of GI-7-ACA: 88%.

[0109] Sum of 3-deacetylation products in the starting Ceph C solution:3% relative to Ceph C

[0110] Sum of 3-deacetylation products in reaction solution obtained: 6%relative to starting Ceph C

EXAMPLE 3

[0111] A cell suspension obtained as described in example 1 ishomogenised before PMSF treatment (DAO activity in homogenised cells:385 U oxidase) and the homogenised cells obtained are treated with PMSFas described in example 1 (DAO activity in homogenised and PMSF treatedcells: 372 U oxidase corresponding to 96% of activity before PMSFtreatment). The cell suspension obtained is reacted with Ceph C asdescribed in example 1.

[0112] Yield of GI-7-ACA: 93%

[0113] Sum of 3-deacetylation products in starting Ceph C solution: 3%relative to Ceph C

[0114] Sum of 3-deacetylation products in reaction solution obtained: 3%relative to starting Ceph C

[0115] Example 3 is repeated but using a DAO activity without PMSFtreatment.

[0116] Yield of GI-7-ACA: 89%.

[0117] Sum of 3-deacetylation products in starting Ceph C solution: 3%relative to Ceph C

[0118] Sum of 3-deacetylation products in reaction solution obtained: 7%relative to starting Ceph C

EXAMPLE 4

[0119] 10 g of a cell pellet obtained as described in example 1 beforecaustic soda treatment are suspended in 50 ml of water, 2 g ofpolyethylene imine (molecular weight 600,000-1,000,000) are added to thesupension obtained and the mixture obtained is incubated for 90 minutesat pH 11 (adjusted by caustic soda solution addition) at roomtemperature. The pH of a mixture obtained is adjusted to 8.5 by additionof phosphoric acid, and the suspension obtained is stirred with 50 ml oftoluene. An emulsion of fine droplet-like cell particles in solvent isobtained to which 8 ml of 25% GDA solution are added. The finedroplet-like cell particles solidify to form solid spherical particles.The solid spherical particles are isolated and washed with spring water.

[0120] DAO activity in spherical particles: 290 U oxidase

[0121] The solid spherical particles obtained are suspended in 50 ml of20 MM phosphate buffer pH 7.0, mixed with 500 μl of PMSF solutionanalogously as described in example 1 and the mixture obtained isincubated for 180 minutes. DAO activity in PMSF treated solid sphericalparticles:

[0122] 305 U oxidase.

[0123] The solid PMSF treated spherical particles obtained are used in aCeph C reaction analogously as described in example 2.

[0124] Yield of GI-7-ACA: 92%

[0125] Sum of 3-deacetylation products in starting Ceph C solution: 3%relative to Ceph C

[0126] Sum of 3-deacetylation products in reaction solution obtained: 3%relative to starting Ceph C

[0127] Example 4 is repeated but using solid spherical particles withoutPMSF treatment.

[0128] Yield of GI-7-ACA: 89%.

[0129] Sum of 3-deacetylation products in starting Ceph C solution: 3%relative to Ceph C

[0130] Sum of 3-deacetylation products in reaction solution obtained: 7%relative to starting Ceph C

EXAMPLE 5

[0131] 10 g of a moist cell pellet of the recombinant E. coli strain,e.g. CCM 4229 (GAC activity: 720 U acylase) isolated from thefermentation broth are washed and re-suspended in 50 ml of 50 mMphosphate buffer pH 7.0. To the suspension obtained 0.5 ml of anethanolic solution containing 10 mg PMSF/ml, are added and the mixtureobtained is stirred for ca. 3 hours at room temperature. GAC activity:708 U acylase (98% of activity before PMSF treatment).

[0132] 10 g of GI-7-ACA are dissolved in 1000 ml of water and the pH isadjusted to 8.2. The PMSF-treated E. coli suspension obtained asdescribed above is added and the mixture obtained is stirred for ca. 180minutes at 12° whilst maintaining pH at ca. 8.0-8.2.

[0133] Yield of 7-ACA: 93%

[0134] Sum of 3-deacetylation products in starting GI-7-ACA solution: 3%relative to GI-7-ACA

[0135] Sum of 3-deacetylation products in reaction solution obtained: 3%relative to starting GI-7-ACA

[0136] Example 5 is repeated but using GAC activity without PMSFtreatment.

[0137] Yield of GI-7-ACA: 90%.

[0138] Sum of 3-deacetylation products in starting GI-7-ACA solution: 3%relative to GI-7-ACA

[0139] Sum of 3-deacetylation products in reaction solution obtained: 7%relative to starting GI-7-ACA

EXAMPLE 6

[0140] 10 g of a moist washed cell pellet of the recombinant E. colistrain, e.g. CCM 4229 (GAC activity: 720 U acylase) are suspended in 50ml of 50 mM phosphate buffer pH 7.0 and homogenised under a pressure of700 bar. The homogenised cells (GAC activity: 752 U acylase) are mixedwith ethanolic PMSF solution as described in example 5. GAC activity inPMSF treated cells: 768 U acylase.

[0141] The suspension obtained is added to a GI-7-ACA solution and thereaction is carried out as described in example 5.

[0142] Yield of 7-ACA: 89%

[0143] Sum of 3-deacetylation products in starting GI-7-ACA solution: 3%relative to GI-7-ACA

[0144] Sum of 3-deacetylation products in reaction solution obtained: 3%relative to starting GI-7-ACA

[0145] Example 6 is repeated but using GAC activity without PMSFtreatment.

[0146] Yield of GI-7-ACA: 83%.

[0147] Sum of 3-deacetylation products in starting GI-7-ACA solution: 3%relative to GI-7-ACA

[0148] Sum of 3-deacetylation products in reaction solution obtained: 6%relative to starting GI-7-ACA

EXAMPLE 7

[0149] Homogenised cells from 10 g of a moist washed cell pellet ofrecombinant E. coli strain, e.g. CCM 4229 cells (GAC activity 7350 Uacylase) are suspended in 50 ml of 50 mM phosphate buffer pH 7.0 andmixed-with a flocculation agent (1 ml of Sedifloc^(R) CL 900-18/40). ThepH of the mixture obtained is adjusted to 5.2 by addition of acetic acidand the mixture obtained is incubated for ca 1 hour at ca. 40° understirring and stirred ca. for a further hour at 10°. The mixture obtainedis cleared by centrifugation and the GAC-containing cell-freesupernatant (GAC activity: 6150 U acylase) is adjusted to pH 7.5, mixedwith PMSF solution and incubated analogously as described in example 5.GAC activity of the cell-free extract obtained after PMSF treatment:6220 U acylase. The mixture obtained is mixed with 10 g of acrylic(immobilisation) beads (Eupergit^(R) C), treated with 50 ml of anaqueous 1 mol sodium sulphate solution and incubated for 64 hours atroom temperature under shaking. The enzyme-charged carrier materialobtained is separated from the immobilisation solution and used ascatalyst in a reaction with a GI-7-ACA solution analogously as describedin example 5.

[0150] Yield of 7-ACA: 90%

[0151] Sum of 3-deacetylation products in starting GI-7-ACA solution: 3%relative to GI-7-ACA

[0152] Sum of 3-deacetylation products in reaction solution obtained: 3%relative to starting GI-7-ACA

[0153] Example 7 is repeated but using immobilised GAC activity withoutPMSF treatment.

[0154] Yield of GI-7-ACA: 83%.

[0155] Sum of 3-deacetylation products in starting GI-7-

[0156] ACA solution: 3% relative to GI-7-ACA

[0157] Sum of 3-deacetylation products in reaction solution obtained: 6%relative to starting GI-7-ACA

EXAMPLE 8

[0158] GAC activity is immobilised analogously as described in example7, but PMSF treatment is not carried out after immobilisation but duringimmobilisation by addition of 1 ml of PMSF solution analogously asdescribed in example S to a mixture containing the GAC-containingcell-free supernatant, Eupergit and sodium sulphate.

[0159] The enzyme-charged PMSF treated carrier material obtained isseparated from the immobilisation solution and used as a catalyst in areaction with a GI-7-ACA solution analogously as described in example 5.

[0160] Yield of 7-ACA and deacetylation products are substantially asdescribed in example 7.

EXAMPLE 9

[0161] GAC-containing supernatant is produced analogously as describedin example 7, but without PMSF treatment (GAC activity: 5830 U acylase).The solution obtained is mixed with 40 g of an ion exchange resin(Relite^(R) Diaion-HPA25L), the mixture obtained is adjusted to pH 7.5and incubated for ca. 300 minutes at room temperature under shaking. Theion exchange resin obtained is isolated and washed (GAC activity: 1865 Uacylase).

[0162] The exchange resin obtained is suspended in 200 ml of 50 mMphosphate buffer, 2 ml of the PMSF solution as used in example 5 areadded and the mixture obtained is incubated for ca. 180 minutes understirring. GAC activity in the PMSF treated ion exchange resin: 1810 Uacylase corresponding to 97% of the activity in the catalyst before PMSFtreatment.

[0163] The enzyme-charged PMSF treated ion exchange resin obtained isused as a catalyst in a reaction with a GI-7-ACA solution analogously asdescribed in example 5.

[0164] Sum of 3-deacetylation products in starting GI-7-ACA solution: 3%relative to GI-7-ACA

[0165] Sum of 3-deacetylation products in reaction solution obtained: 3%relative to starting GI-7-ACA

EXAMPLE 10

[0166] a. Pre-Treatment

[0167] Cells of Trigonopsis variabilis, e.g. ATCC 58536 (535 g moistweight, DAO activity: 25,970 U oxidase) are pre-treated in 2.7 kg of afermentation broth by stirring 21 g of an aqueous 25% GDA solution intothe fermentation broth during ca. 1 hour at room temperature and at pH7.5. The cells are harvested from the cell suspension obtained bycentrifugation and stored in a frozen state. The frozen cell mass isthawed, washed with spring water by means of micro-filtration andconcentrated to a volume of ca. 735 ml. DAO activity in the concentratedcell mass: 26,350 U.

[0168] b. Polymer Treatment

[0169] 735 ml of the cell mass obtained as described in step a. (100 gcell dry weight) are suspended with mercaptoethanol (5 mM), EDTA (2 mM)and 40 g of an aqueous 50% PEI solution, molecular weight600,000-1,000,000 in water (ca. 800 ml total volume) and stirred slowlyfor ca. 90 minutes at pH 11 which is adjusted by addition of a causticsoda solution at room temperature. The pH of the mixture obtained isadjusted to pH 9 by addition of phosphoric acid. DAO activity: 24,360 U(94% of pre-treated DAO activity obtained as described in example 10 a.

[0170] c. Crosslinking

[0171] To 3000 ml of TBP in a stirring vessel the cell/polymer mixtureobtained in step b. is added at room temperature under stirring. Ahomogeneous dispersion of the cell/polymer droplets in TBP is obtainedwithin ca. 30 minutes to which 40 g of an aqueous 25% GDA solution areadded. Cosslinking starts immediately and is terminated after someminutes and the cells are obtained in the form of solid sphericalparticles. 2000 g of glycerol are added to the mixture obtained at 15°to 20° and phase separation between the TBP phase and the aqueous phaseis achieved. The lower phase containing mainly glycerol and solidspherical cell particles is separated from the upper phase containingmainly TBP. The upper phase obtained (2900 ml) may be re-used e.g. inanother crosslinking batch. The solid spherical particles are isolatedfrom the lower phase after ca. 90 minutes and washed with spring waterin order to remove TBP and glycerol residues.

[0172] 580 g of moist solid spherical particles having oxidase activityare obtained, corresponding to 121 g of dry weight. Specific DAOactivity in the solid spherical particles is 32 U/g moist weight (153U/g dry weight), i.e. total DAO activity is 18,560 U (71% of thepre-treated DAO activity obtained as described in example 10 a.

EXAMPLE 11

[0173] 580 g of the solid spherical particles (moist weight) obtained inexample 10 c. are suspended in 2000 ml of spring water and stirred atroom temperature. 20 mg of PMSF are dissolved in 20 ml of absoluteethanol and the solution obtained is added to the oxidase suspensionwithin ca. 2 minutes. The mixture is incubated for ca. 3 hours, thesolid spherical particles obtained are isolated and washed with aqueousethanol (1000 ml, 10%) and with spring water.

[0174] 563 g of solid spherical particles (moist weight; correspondingto 118 g of dry weight) are obtained. Specific DAO activity in the PMSFtreated solid spherical particles is 31 U/g moist weight (148 U/g dryweight), i.e. total DAO activity is 17,450 U (67% of the pre-treated DAOactivity obtained as described in example 10 a. and 94% of the activitybefore PMSF treatment). The esterase activity in the spherical particlesobtained is substantially removed.

EXAMPLE 12

[0175] The pH of a cell/polymer mixture produced as described inexamples 10 a. and b. is adjusted to 9.0 by addition of phosphoric acidand 80 g of aluminium oxide are added. Crosslinking is carried out withthe mixture obtained as described in example 10 c.

[0176] 630 g of solid spherical particles (moist weight, correspondingto 193 g of dry weight) are obtained. Specific DAO activity in the solidspherical particles is 26 U/g moist weight (85 U/g dry weight), i.e.total DAO activity is 16,440 U (63% of the pre-treated DAO activityobtained as described in example 10 a.). A narrowsize-distribution-range of the particles of 240-500 μm is obtained.

EXAMPLE 13

[0177] Is carried out analogously as described in example 10 but withoutaddition of glycerol. After crosslinking the suspension is stirred forca. 60 minutes. The solid spherical particles obtained in TBP suspensionare isolated by running the suspension obtained through a vessel havinga perforated bottom, wherein the perforation holes are smaller than thesize of the solid spherical particles obtained. 2680 ml of TBP areobtained. TBP residues are removed from the solid spherical particlesobtained by washing with spring water.

[0178] 610 g of solid spherical particles (moist weight, correspondingto 126 g of dry weight) are obtained. Specific DAO activity in the solidspherical particles is 36 U/g moist weight (174 U/g dry weight), i.e.total activity is 21,960 U (85% of the pre-treated DAO activity obtainedas described in example 10 a.

EXAMPLE 14

[0179] PMSF treated solid spherical particles obtained analogously asdescribed in examples 10 and 11 (81 g moist weight, DAO activity: 2,500U) are mixed with 1 liter of a 75 mM aqueous Ceph C solution of pH 7.2.Air under 5 bar pressure is introduced into the mixture obtained for ca.80 minutes. The solution is separated off from the solid sphericalparticles and is analysed by HPLC. 0.6 MM Ceph C, 70.7 mM GI-ACA and 3mM of KA-7-ACA are determined in the separated solution.

[0180] Example 14 is repeated 142 times each time using a fresh CephC-solution and each time using the same PMSF treated solid sphericalparticles described above in example 14.

[0181] The solution is separated off from the solid particles in batch142 after ca. 160 minutes of contact with Ceph C and is analysed byHPLC. 0.5 mM Ceph C, 69.8 mM GI-ACA and 0.4 mM of KA-7ACA are determinedin the separated solution.

[0182] After batch 142 the solid spherical particles had been used for308 hours. 93 g of solid spherical particles (moist weight,corresponding to 24 g of dry weight) are isolated.

[0183] Specific DAO activity in the solid spherical particles obtainedis 12 U/g moist weight corresponding to 47 U/g dry weight, i.e. totalactivity is 1,120 U (45% of the original activity).

EXAMPLE 15

[0184] Cells of Schizosaccharomyces pombe, e.g. ATCC 38399 and e.g. ATCC38436 (82 g and 90 g moist weight, respectively) having DAO activity areeach pre-treated (separately) analogously as described in example 10 a.with 3.3 g of an aqueous 25% GDA solution and harvested and stored in afrozen state. After thawing the mixture is washed with spring water andconcentrated to obtain 20.4 g of dry substance in 164 ml from e.g. ATCC38399 and 24.1 g of dry substance in 180 ml from e.g. ATCC 38436.

[0185] 7 g of cell dry weight of each of e.g. ATCC 38399 and e.g. ATCC38436 are treated (separately) with 2.8 g of an aqueous 50% PEI solutionas described in example 10 b.

[0186] Crosslinking is carried out with both suspensions obtained(separately) analogously as described in example 10 c. in 230 ml TBPunder addition of 3.5 g of an aqueous 25% GDA solution and underaddition of 400 g of glycerol. The solid spherical particles obtainedare washed with spring water over a sieve in order to remove glyceroland TBP residues.

[0187] 59.5 g moist weight of solid spherical particles having DAOactivity (14.0 g dry weight) are obtained from e.g. ATCC 38436 and 55.6g moist weight of solid spherical particles having DAO activity (16.1 gdry weight) are obtained from e.g. ATCC 38399. The solid sphericalparticles obtained show similar DAO reaction characteristics and similarstability characteristics as the spherical particles obtained asdescribed in example 10 c.

[0188]Schizosaccharomyces pombe is suitable for the cloning andexpression of various enzymes. Other biocatalysts, e.g. solid sphericalparticles having other enzyme activity may be obtained in a simplemanner analogously as described in the present example.

EXAMPLE 16

[0189] Cells of Pleurotus ostreatus (90 g moist weight, 18,970 Upenicillin V acylase) are pretreated with 3.3 g of an aqueous 25% GDAsolution analogously as described in example 10 a. The cells from thecell suspension obtained are harvested, stored in a frozen state, andafter thawing are washed with spring water and concentrated bymicrofiltration.

[0190] 18.6 g of cells obtained (dry weight) are treated with 7.4 g ofan aqueous 50% PEI solution. Cell crosslinking is carried outanalogously as described in example 10 c. in 560 ml TBP under additionof 7.4 g of an aqueous 25% GDA solution and under addition of 370 g ofglycerol. The solid spherical particles obtained are washed with springwater through a sieve in order to remove glycerol and TBP residues.

[0191] 223 g of solid spherical particles containing penicillin Vacylase (moist weight corresponding to 29.4 g dry weight) are obtained.

[0192] Specific penicillin V acylase is 50.4 U/g moist weight, i.e.total penicillin V acylase is 11,240 U (59% the original activity).

[0193] 1 U penicillin V acylase corresponds to the formation of 1 μmolper minute of 6-aminopenicillanic acid in an aqueous phosphate-bufferedK-penicillin-V solution (50 g/l) at pH 7.5 and 28°.

EXAMPLE 17

[0194] Cells of Trigonopsis variabilis, e.g. ATCC 58536 (54 g moistweight, DAO activity: 2,600 U oxidase) are produced analogously asdescribed in examples 10 a. and 10 b.

[0195] Crosslinking is effected analogously as described in example 10c., but a cell/polymer mixture containing Trigonopsis variabilis cells(ca. 10 g dry weight) is added under stirring into either 300 ml ofglutaric dimethylester or 300 ml of adipic dimethylester (instead of3000 ml of TBP to 100 g cell dry weight). Crosslinking in each of themixtures obtained (separately) is carried out under addition of 5 g ofan aqueous 25% GDA solution to each of the mixtures and under additionof 400 g of glycerol to each of the mixture at a temperature of 15° to20° analogously as described in example 10c.

[0196] 93.4 g and 86.9 g, respectively of solid spherical particleshaving DAO activity are obtained (moist weight, corresponding to 20.6 gand 20.2 g of dry weight, respectively).

[0197] Specific DAO activity is 14.8 U/g moist weight or 12.5 U/g moistweight, respectively, i.e. total activity is 1382 U (53% of the originalactivity) or 1086 U (42% of the original activity), respectively.

1. A process for decreasing esterase activity present in the presence ofD-amino acid oxidase activity or in the presence of glutarylacylaseactivity in a mixture having esterase activity and Damino acid oxidaseactivity and/or glutarylacylase activity comprising treating a mixturehaving esterase activity and D-amino acid oxidase activity and/orglutarylacylase activity with phenylmethylsulphonyl fluoride.
 2. Aprocess for the production of glutaryl-7-aminocephalosporanic acidcomprising the steps i) treating a mixture having esterase activity andD-amino acid oxidase activity with phenylmethylsulphonyl fluoride, ii)reacting cephalosporin C with a mixture obtained in step i) to obtainglutaryl-7-aminocephalosporanic acid; and isolatingglutaryl-7-aminocephalosporanic acid obtained, if desired.
 3. A processfor the production of 7-aminocephalosporanic acid comprising the stepsi) treating a mixture having esterase activity and glutarylacylaseactivity with phenylmethylsulphonyl fluoride, ii) reactingglutaryl-7-aminocephalosporanic acid with a mixture obtained in step i)to obtain 7-aminocephalosporanic acid; and isolating7-aminocephalosporanic acid obtained in step ii), if desired.
 4. Aprocess for the production of spherical particles from microogranismcells having an enzyme activity, comprising the steps i) treatingmicroorganism cells with a primary or secondary amine containingpolymer, ii) contacting an organic solvent which is able to form atwo-phase system with water with a mixture of microorganism cells, iii)treating a mixture obtained in step ii) with a bifunctional agent; andisolating the spherical particles obtained.
 5. Spherical particlescontaining microorganism cells which contain enzyme activity obtainablefrom a process comprising the steps i) treating microorganism cellshaving enzyme activity with a primary or secondary amine containingpolymer ii) contacting an organic solvent which is able to form atwo-phase system with water with a mixture of microorganism cells, iii)treating a mixture obtained in step ii) with a bifunctional agent; andisolating the spherical particles obtained.
 6. Spherical particlesobtained by a process comprising the steps as defined in claim
 5. 7. Aprocess for the production of glutaryl-7-aminocephalosporanic acidcomprising the steps i) treating microorganism cells having D-amino acidoxidase activity with a primary or secondary amine containing polymer,ii) contacting an organic solvent which is able to form a two-phasesystem with water with a mixture of microorganism cells, iii) treating amixture obtained in step ii) with a bifunctional agent; and isolatingthe spherical particles obtained, and if desired, treating a mixturehaving esterase activity in the presence of D-amino acid oxidaseactivity with phenylmethylsulphonyl fluoride, or treating sphericalparticles obtained in step iii) having esterase activity in the presenceof D-amino acid oxidase activity with phenylmethylsulphonyl fluoride,iv) reacting cephalosporin C with spherical particles obtained in stepiii) to obtain glutaryl-7-aminocephalosporanic acid, and isolatingglutaryl-7-aminocephalosporanic acid, if desired.
 8. A process for theproduction of glutaryl-7-aminocephalosporanic acid which comprisesreacting cephalosporin C with spherical particles obtainable by stepiii) as defined in claim 7 to obtain glutaryl-7-aminocephalosporanicacid, and isolating glutaryl-7-aminocephalosporanic acid, if desired. 9.A process for the production of 7-aminocephalosporanic acid whereinglutaryl-7-aminocephalosporanic acid obtained according to claim 2, stepii) or claim 7, step iv) is converted into 7-aminocephalosporanic acid.10. Use of a process as claimed in claim 4 or use of spherical particlesas claimed in claim 5 in the production of7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid from cephalosporin Cor from glutaryl-7-aminocephalosporanic acid.
 11. A process for theproduction of 7-aminocephalosporanic acid comprising the steps i)treating microorganism cells having glutarylacylase activity with aprimary or secondary amine containing polymer, ii) contacting an organicsolvent which is able to form a two-phase system with water with amixture of microorganism cells, iii) treating a mixture obtained in stepii) with a bifunctional agent; and isolating the spherical particlesobtained, iv) if desired, treating a mixture having esterase activity inthe presence of glutarylacylase activity with phenylmethylsulphonylfluoride, or treating spherical particles obtained in step iii) havingesterase activity in the presence of glutarylacylase acylase withphenylmethylsulphonyl fluoride, if desired, v) reactingglutaryl-7-aminocephalosporanic acid with spherical particles obtainedin step iii) or obtained in step iv) to obtain 7-aminocephalosporanicacid; and isolating 7-aminocephalosporanic acid obtained, if desired.12. A process for the production of a cephalosporin wherein7-aminocephalo-sporanic acid or7-amino-3-hydroxymethyl-3-cephem-4-carboxylic acid produced according tothe present invention according to any one of claims 3 or 9 is used asan intermediate.
 13. Product produced by a process according to any ofclaims 1 to 4, 7 to 9 or 11 to 12.